Pathogenic bacteria make extensive use of secreted proteins in order to colonize and persist in their hosts. Recently, a type VI protein secretion system (T6SS) of Burkholderia pseudomallei, the causative agent of melioidosis, was shown to be a major virulence determinant of the organism. The mechanistic basis for the role of T6S in B. pseudomallei whence is not known. Interestingly, the genome of this organism encodes five additional T6SSs that have not been investigated; accounting for greater than 2% of its genomic coding capacity. Given the widespread relevance of this protein translocation system in bacteria-host interactions, it is likely that these additional T6SSs also play critical roles for this organism. Having discovered T6S in the closely related organism Pseudomonas aeruginosa, the project leader of this proposal, Dr. Joseph Mougous, has demonstrated expertise in this field of research. The focus of this proposal is to characterize several aspects of the T6SSs of B. pseudomallei. In Aim 1, the involvement of each system in virulence and host interactions will be measured. Aim 2 will identify regulatory pathways of the systems, and investigate the role of a predicted post-translational control mechanism. In the final aim, novel T6S substrates will be identified by a quantitative mass spectrometric approach. The phenotypic profile of each secretion system, the identification of regulators, and the identities of T6S substrates, will provide a solid foundation for future investigation of T6S in this organism.